R A Leng*, Sangkhom Inthapanya and T R Preston**

Abstract

Three experiments were carried out to evaluate
the effect of biochar on methane production from buffered ruminal fluid in an
in vitro system using cassava root meal as substrate with either potassium
nitrate or urea as the NPN source.

Experiment 1: The treatments in 2*2 factorial
arrangements with four replications of each treatment were: urea or potassium
nitrate as NPN source; and presence or absence of 5% biochar. The quantity of
substrate was 12 g DM to which was added 240 ml rumen fluid (from slaughtered
buffalo) and 960 ml of buffer solution. The incubation was for 24 and 48hours
with measurements of gas production, percent methane, substrate solubilized and
methane produced per unit substrate solubilized.

Gas production, methane percentage in the gas,
substrate solubilized and methane produced per unit substrate solubilized were
all lowered when nitrate replaced urea as the fermentable N source at either 24
or 48 hours of the incubation. Addition of biochar did not affect gas production
but increased the percentage DM solubilized. Methane produced and methane
produced per unit substrate solubilized was lowered by 14% due to addition of
biochar when urea was the NPN source but was not affected when nitrate was the
source of NPN.

Experiment 2: The treatments in a 2*6 factorial
with three replications were: (i) concentration of biochar (0, 1, 2, 3, 4 and 5%
on DM basis); (ii) washing or no washing of the biochar. The substrate was
cassava root meal and urea. The general procedure and analyses were similar to
those in experiment 1.

Methane produced was reduced by 11-13% by adding
1% biochar but there were no further benefits from increasing the biochar level
to between 2 and 5%. Methane production and per unit substrate DM solubilized
were reduced by about 5% by washed compared with unwashed biochar.

Biochar at 0.5% reduced methane by 10% and at 1%
reduced it by 12.7%. With 50% nitrate N and 50% urea N, plus biochar at 1%,
the reduction in methane was 40.5% and with 100% nitrate N plus biochar at
1%, it was 49%.

Introduction

Methane emissions from biological sources are a balance between production by
methanogenic Archae and oxidation by methanotrophic micro-organisms.
Methane oxidation has been reported in both aerobic and anaerobic environments,
which restricts the flux of methane entering the atmosphere (Hanson and Hanson
1996). Measurements in flooded rice fields indicated that a high proportion (up
to 80%) of the methane produced was oxidized at the soil surface (Hanson and
Hanson 1996). Both aerobic and anaerobic methanotrophic bacteria are unique in
their ability to utilize methane as a sole carbon and energy source.

Stocks and McCleskey (1964) isolated methane-utilizing bacteria from the rumen of steers
that were similar to methanotrophs isolated from soil and water and Mitsumori et
al (2002) demonstrated methanotrophs were present in both rumen fluid and
attached to the rumen wall. However, a study using an artificial rumen indicated
that only 0.3% of methane flux was oxidized (Kajikawa and Newbold 2000). Further
studies by Kajikawa et al (2003) indicated that 0.2 to 0.5 of the methane flux
was anaerobically oxidized by reversal of methanogenesis with sulphate as the
terminal electron acceptor.

Recent studies have demonstrated that the application of biochar in paddy soils
lowered methane release (Liu et al 2011) although other studies under different
conditions report the opposite (Zhanga et al 2010). It appears that amounts of
methane emissions will depend on the soil type, the chemical properties of the
biochar, and on the fertilization and water management regimes (Cai et al 1997).
However the decrease in methane emissions under biochar amendment in the
research of Liu et al (2011) was not the result of inhibition of the growth of
methanogenic archaea but to increased methanotrophic proteobacterial abundances
with greatly increased ratios of methanotrophic to methanogenic abundances in
the paddy soils as measured by real-time polymerase chain reaction (qPCR) and
PCR–DGGE (denaturing gradient gel electrophoresis) (Feng et al 2012). The
possibility of increasing methanotrophic activity in the rumen led us to examine
the effect of biochar amendment on ruminal fluid methane production in vitro
as a preliminary to whole animal research.

It is now
well established that nitrate
can replace urea as a major source of fermentable N (rumen
ammonia) in rations fed to ruminants (see Trinh Phuc
Hao
et al 2009)
and at the same time
it lowers
methane
production because
of it’s higher affinity for hydrogen as compared to carbon dioxide (see Leng
2008). In
preliminary studies the methane mitigating effect of biochar was seen and it was therefore
decided to examine the possibility of an additive effect of nitrate and biochar.

To test the effect of biochar on methane production from
buffered ruminal fluid in an in vitro system using cassava root meal as
substrate and with either potassium nitrate or urea as the NPN source.

Materials and methods

Three in vitro incubation experiments were conducted in the laboratory of
the Faculty of Agriculture and Forest Resources, Souphanouvong University, Luang
Prabang province, Lao PDR, from April to May 2012.

The substrate was put in the incubation flask containing the diluted rumen fluid
which was then gassed with carbon dioxide and the flasks were incubated at 38
0C in a water bath for 24 and 48 hours.

Data
collection and measurements

The gas volume was read from the collection bottles
directly after 24 and 48 hours and the percentage of methane in the gas
was measured using a Crowcon infra-red analyser (Crowcon Instruments Ltd, UK)
for the separate incubations. Gas from the collection bottle was drawn into the measuring apparatus. Three samples were
measured from each collection bottle.At the end of each incubation time the residual
insoluble substrate in the incubation bottle was determined by filtering the
contents through several layers of cloth that
retained particle sizes to at least 0.1mm and then this was dried (100°C for 24
hours) and weighed.

The data were analyzed by the General Linear Model (GLM)
option in the ANOVA program of the Minitab (2000) Software. Sources of variation
in the model were: Biochar, NPN source, interaction Biochar*NPN and error.

Experiment 2

The objectives were to examine the effect of the concentration of biochar in the
fermentation medium over the range of 0 to 5% (DM basis).

The design was a 2*6 factorial with three replications. The factors were:

Washed or unwashed biochar

Concentration of biochar (0, 1, 2, 3, 4 or 5% on DM basis)

The general procedure and analyses were similar to those in experiment 1.

Biochar was used either as an untreated powder or after washing. Biochar was
washed by adding 100ml of distilled water to 50g of biochar, stirring and the
flask allowed to stand for 10 minutes before pouring off the water phase. This
was repeated two times and the biochar dried before adding to the
incubation flasks containing buffer, rumen fluid and substrate.

Statistical analysis

The data were analyzed by the General Linear Model (GLM)
option in the ANOVA program of the Minitab (2000) Software. Two sets of data
were analysed. The first set included all the data to test effect of levels of biochar.
The sources of variation were: levels of biochar and error.
In the second set the data for the control treatment were omitted so as to test
the effect of washing or not washing the biochar. Sources of variation in the
model for set 2 were: Level of biochar, washed or not washed, interaction level of biochar*washing
and error.

Experiment 3

Objectives and experimental design

The objectives were to examine a lower concentration of biochar (0.5% in
substrate DM) and the interaction with different proportions of potassium
nitrate and urea as NPN source in diluted rumen fluid incubated with cassava
root meal as described above .

The design was a completely randomized comparison of:

No biochar with urea (2% in DM)

0.5% biochar with urea (2% in
DM)

1.0% biochar with urea(2% in DM)

1.0% biochar with 50% urea (1%
in DM) and 50% nitrate (3% in DM)

1.0% biochar with 100% nitrate
(6% nitrate in DM)

There
were 4 replicates of each treatment. The general procedure and analyses were
similar to those in experiment 1.

Statistical analysis

The data were analyzed by the General Linear Model (GLM)
option in the ANOVA program of the Minitab (2000) Software. Sources of variation
in the model were: Treatments and error.

Effects of Biochar

Addition of biochar did not affect gas production but increased the percentage
DM solubilized. Methane produced and methane produced per unit substrate
solubilized were lowered by added biochar by 11.5 and 12.8% at 24 and 48h when urea was the NPN source
but appeared to be less affected (interactions were P=0.16 and P=0.23 at 24h and
P=0.052 and P=0.069 at 48h) when nitrate was the source of NPN (8.3
and 11.5%; Figures 1 to 4). These effects (for lowering of methane production)
were thus similar for incubation periods
of 24 and 48 h with urea, but more pronounced for the longer period of fermentation (Table
2).

Table 2. Mean values of gas
production, methane percentage in the gas, DM solubilized and
methane production per unit substrate solubilized after 24 or 48 h
of fermentation, in in vitro fermentation of cassava root meal
supplemented with potassium nitrate or urea,and with or without
addition of biochar (gas
production has been corrected for the carbon dioxide that would be
released when the urea was hydrolyzed)

Biochar

No Biochar

Prob.

K-nitrate

Urea

SEM

Prob.

P (B*NPN)

0-24 hours

Gas
production, ml

Total

1206

1171

0.368

878

1500

26.43

<0.001

0.794

Corrected#

1148

1113

0.368

878

1384

26.43

<0.001

0.794

Methane, %

9.00

10.4

<0.001

7.00

12.4

0.198

<0.001

0.205

Methane, ml

117

130

0.033

61.3

186

4.103

<0.001

0.161

Digested, %

60.5

59.0

0.037

57.1

62.5

0.451

<0.001

0.489

Methane, ml/g DM substrate

16.3

18.6

0.024

9.51

25.3

0.635

<0.001

0.226

0-48 hours

Gas
production, ml

Total

1625

1669

0.343

1469

1825

31.35

<0.001

0.229

Corrected#

1567

1611

0.343

1469

1707

31.35

<0.001

0.229

Methane, %

13.0

14.4

0.019

8.25

19.1

0.357

<0.001

0.472

Methane, ml

219

251

0.003

121

349

6.287

<0.001

0.052

Digested, %

63.1

62.2

0.035

59.7

65.6

0.276

<0.001

0.471

Methane, ml/g DM substrate

29.2

34.0

0.002

18.0

45.3

0.856

<0.001

0.069

# Corrected by subtracting CO2 derived from urea

Figure 1.Effect of biochar and potassium
nitrate or urea as NPN source on methane production after 24 hours
of fermentation

Figure 2.Effect of biochar and potassium
nitrate or urea as NPN source on methane production after 48 hours
of fermentation

Figure 3. Effect of
biochar and potassium nitrate or urea as NPN source on methane
production per unit of substrate DM solubilized after 24 hours of
fermentation

Figure 4. Effect of
biochar and potassium nitrate or urea as NPN source on methane
production per unit substrate DM solubilized after 48 hours of
fermentation

Experiment 2

Effects of
level of biochar

Methane produced in 24h was reduced by adding 1% biochar (by 12%) but there was
no further effects from raising biochar level to between 2 and 5% (Table 3;
Figure 5). The same effect was observed when methane production was expressed as
ml per unit substrate DM solubilized (Figure 7).

Effect of washing the biochar

Methane production in 24h and per unit substrate DM solubilized was reduced by
about 5% by washing the biochar (Table 3; Figures 6 and 8).

Figure 5.
Effect of level of biochar on methane production after 24
hours of fermentation

Figure 6.
Effect of washing the biochar on methane production after 24 hours
of fermentation

Figure 7.
Effect of level of biochar on methane production per unit of
substrate DM solubilized after 24 hours of fermentation

Figure 8.
Effect of washing the biochar on methane production per unit
of substrate DM solubilized after 24 hours of fermentation

Experiment 3

Biochar at 0.5% of the substrate reduced methane by 10% and at 1% reduced it by
12.7% (Table 4; Figures 9 and 10). With a 50% mix of nitrate and urea N,
plus biochar at 1%, the reduction in methane was 40.5% and with 100% nitrate N
plus biochar at 1%, it was 49%.

Figure 9.
Effect of level of biochar (0, 0.5 or 1%) with urea or 1%
biochar with combinations of urea and nitrate, on methane
production after 24 hours of fermentation

Figure 10.
Effect of level of biochar (0, 0.5 or 1%) with urea, or 1% biochar
with combinations of urea and nitrate, on methane production per unit DM solubilized after 24 hours of fermentation

Discussion

As has been observed in numerous studies nitrate lowers methane production from
rumen fluid indicating the presence of nitrate reducing bacteria that use
nitrate as a terminal electron acceptor and outcompete methanogens for hydrogen
produced in fermentation. However this is the first study to show that biochar
may also have a role in reducing rumen methanogenesis. Adding 5% biochar to the
substrate used in the incubation flasks apparently lowered net methane production by
14% and by 13% when this was calculated on an, as is or, per unit of dry matter
apparently fermented basis, respectively. In the presence of nitrate there was a
significant reduction of methane production by 34 %. The presence of biochar
appeared to further lower methane production in the presence of nitrate but the
lowered production was quantitatively small but still 9% of the methane produced
when nitrate was incubated without biochar.

In the second study increasing levels of biochar added to the incubation medium
demonstrated that the optimum biochar level for maximum mitigation in vitro
was
less than 1% of the substrate added. Even though the major reduction in
methane production by biochar was similar to that in the first study, at every
level of inclusion of unwashed biochar in the incubation medium there was both a
higher total methane production and methane production per unit of
substrate apparently digested.So washing the biochar appears to increase it’s
methane mitigating benefits.Biochar’s overall mechanism for lowering methane
production appeared to be mostly associated with its insoluble components.

This research was initiated because of the demonstration that biochar may
encourage methanotrophic microbes to proliferate in anoxic soils associated with
rice growing (Feng et al 2012). However it is recognized that biochar does not
always decrease methane release from amended soils (Cai et al 1997) and there
are multiple interactions that come into play in any anoxic ecosystem that may
affect the results. Similarly it will be necessary to study different biochars
with differing sources of rumen fluid to clarify the potential mitigation
possibilities for enteric methane.

It appears unlikely that biochar lowers methane production by anaerobic
oxidation as in natural anaerobic environments methanotrophs grow slowly
limited by the energy availability. The short turn over rate of rumen fluid
appears to negate
the substantial growth of these organisms. However methanotrophs depend on methane
oxidation and they are also sulphur-reducing bacteria (SRB) because
sulphur is the terminal electron acceptor in anaerobic methane oxidation
according to the equation CH4+SO42− ->HCO3−
+HS−+H2O. Other studies have suggested that SRB did not
carry out anaerobic oxidation directly, but rather a consortium with unknown
organisms and SRB was involved (Smemo and Yavitt 2011). However, it appears
that anaerobic methanotrophs are present in rumen fluid (Stock and
McCleskey 1964; Kajikawa et al 2003)
and aerobic methanotrophs are attached to rumen epithelium (Mitsumori
et al 2002). To be effective in capturing methane, anaerobic methanotrophs would
have to be spatially distributed close to the site of methane production which
is likely to be close to the methanogenic Archae that probably colonize
the outer layers of the biofilm consortia attached to feed particles (McAllister
and Cheng
1996; Leng 2011) where the partial pressure of methane will be the highest.

The spatial distribution of organisms relative to their preferred substrate is
important as illustrated in in vitro incubations of marine
sediments containing high numbers of microbial consortia, consisting of
organisms that affiliate with methanogenic archaea and with sulphate-reducing
bacteria, where an increase in partial pressure of the methane from 0.1 MPa
(approximately 1 atm) to 1.1 MPa (approximately 11 atm) resulted in a four to
fivefold increase of the sulphide production rate and therefore methane
oxidation (Nauhauset al 2002), Thus the spatial distribution of methane oxidizing
organisms or consortia close to the site of methanogenesis appears to be a
critical issue in stimulating the overall reactions. The question raised here
is “does biochar with its relatively large surface area (http://en.wikipedia.org/wiki/BET_theory)
and highly porous structure (Photo 1) providea
favourable habitat for the organisms involved in a methanogenic
methanotrophic interaction increasing the potential for anaerobic methane
oxidation”. This then leads to ecological studies of how best to increase
the efficiency of these associations. The BET surface area is a measure of the
ability of a material to absorb gases. Biochars often have BET surface areas of
2-4 m2 /g biochar but much greater surface areas maybe produced by
particular production technologies. As shown in the photo the potential to
create habitat for biofilm residing microbes is substantial where gases could be
adsorbed on to the surfaces of the biochar.

There are also other potential explanations for the net decrease in methane release
from rumen fluid including a change in surface
ion exchange capacity for microbial biofilm
formation or a direct effect of chemicals not soluble in water on fermentation
pathways and the end products produced. A direct toxic effect on methanogens
seems unlikely as the rate of fermentation of the substrate appeared to be
unchanged by biochar addition and the amounts of biochar were exceedingly small.
It is also unlikely that the biochar washed or unwashed could supply high
affinity electron accepting substrate, particularly at the lowest level of
inclusion (1% of the total substrate). However hydrogen uptake could be reduced
in some way as nitrate out-competes most other electron acceptors (sulphate and
carbon dioxide) and the effect of biochar appears to be reduced when nitrate
replaced urea as the major fermentable N source. Nitrate appears to inhibit
anaerobic methane oxidation (Hanson and Hanson 1996). Since the depression in
methane production was observed in vitro there can be no involvement of the
rumen wall associated methanotrophs, which are probably aerobic bacteria
dependant on diffusion of oxygen across the rumen epithelium as the terminal
electron acceptor (Mitsumori et al 2002).

The lack of, or small, response of methane production to biochar inclusion in the substrate
when nitrate provided the fermentable N source may be a result of nitrate
inhibition of methanotrophs through competition with these sulphur reducing bacteria
for available electrons.

The preliminary and speculative nature of the present report is acknowledged but
the importance of this observation to atmospheric methane accumulation if
repeatable in other situations is so immense that bringing the finding to
the attention of other research scientists is warranted and this early
publication is to put the information in the public domain as it is felt that
any attempt to patent a process using biochar to mitigate enteric methane
production from all animals is not in the interests of people in general. If these
results are
repeated when animals are fed biochar in their diet, then there would be
good reason to suggest that enteric methane production maybe lowered from all
animals including humans by extremely small amounts of dietary biochar whether
fermentation sacs precede or follow digestion in the intestines. The exception
will be where acetogenesis replaces methanogenesis in the fermentation areas of
the tract as in the kangaroo that produce little methane (Kempton et al 1976). A major point here is that if the results are applicable to animals under
domesticated conditions the small amounts likely to be needed would probably
indicate this could be the least expensive methodology for mitigating
methane production.

Conclusions

Methane production by rumen fluid was decreased when nitrate
replaced urea as NPN source at both 24 and 48 h of fermentation time.

There was no effect of
added biochar on gas production but the percentage of DM solubilized
was increased.

Methane production and methane per unit substrate solubilized was reduced by added biochar with
urea as NPN source.

Overall, the methane production by rumen fluid was reduced by 12% by adding 1% of biochar and there were no further benefits from increasing biochar content.

Washing the biochar appeared to improve its methane mitigation benefits.

With 3% KN and 1% urea, plus biochar at 1%, the reduction in methane was 40.5% and with 6% KN plus biochar at 1%, it was 49%.

Acknowledgements

The authors acknowledge support for this research from the MEKARN project
financed by Sida. Special thanks to Mr Sengsouly Phongphanith who provided
valuable help in the laboratory and preparation of the samples. Thanks
also to the Department of Animal Science laboratory, Faculty of Agriculture and
Forest Resource, Souphanouvong University for providing the facilities to carry
out this research.

References

Binh Phuong L T, Preston T R and Leng R A
2011 Mitigating methane production
from ruminants; effect of supplementary sulphate and nitrate on methane
production in an in vitro incubation using sugar cane stalk and cassava leaf
meal as substrate. Livestock Research for Rural Development. Volume 23, Article
#22. http://www.lrrd.org/lrrd23/2/phuo23022.htm

Inthapanya S, Preston T R and Leng R A 2011
Mitigating methane production from ruminants; effect of calcium nitrate as
modifier of the fermentation in an in vitro incubation using cassava root as the
energy source and leaves of cassava or Mimosa pigra as source of protein.
Livestock Research for Rural Development. Volume 23, Article #21. http://www.lrrd.org/lrrd23/2/sang23021.htm

Silivong P, Preston T R and Man N V 2012
Effect of supplements of potassium nitrate or urea as sources of NPN on methane
production in an in vitro system using molasses and Paper mulberry or Muntingia
foliages as the substrate. Livestock Research for Rural Development. Volume 24,
Article #69. http://www.lrrd.org/lrrd24/4/sili24069.htm

Thanh V D, Preston T R and Leng R A 2011:
Effect on methane production of supplementing a basal substrate of molasses and
cassava leaf meal with mangosteen peel (Garcinia mangostana) and urea or nitrate
in an in vitro incubation. Livestock Research for Rural Development. Volume 23,
Article #98. http://www.lrrd.org/lrrd23/4/than23098.htm